Characterization and phylogenetic analysis of the complete mitochondrial genome of Saccharomycopsis fibuligera (lindner) Klocker 1907 (saccharomycetales: saccharomycopsidaceae)

Abstract Saccharomycopsis fibuligera (Lindner) Klocker 1907 is frequently employed in the fermentation of metabolites such as citric acid, ethanol, mannitol, and pyruvate. Its heat tolerance and alcohol-producing capabilities during fermentation make it a desirable option for bread and wine production. To date, the mitochondrial genome of S. fibuligera has not been sequenced. In the present study, we obtained the full mitochondrial genome of S. fibuligera, which is 57,302 bp long and has a GC content of 24.40%. This genome contained 14 core protein-coding genes, 3 independent ORFs, 21 intronic ORFs, 25 tRNAs, and 2 rRNA genes. By utilizing the Bayesian inference phylogenetic method, we constructed phylogenetic trees for 24 Saccharomycotina fungi, which indicated that S. fibuligera is closely related to S. capsularis.


Introduction
Saccharomycopsis fibuligera (Lindner) Klocker 1907 is an aerobic yeast species that has garnered attention for its ability to grow on a variety of carbon sources, including glucose, xylose, and glycerol (Chi et al. 2009;Moon et al. 2021).Its tolerance to high osmolarity environments makes it an ideal candidate for biotechnological applications such as the production of biofuels (Favaro et al. 2015).Moreover, S. fibuligera has been widely used in the fermentation of metabolites such as citric acid, ethanol, mannitol, and pyruvate (Su et al. 2020;Yang et al. 2021).Furthermore, its ability to tolerate high temperatures and form alcohols during fermentation makes it a suitable choice for bread and wine making (Lee et al. 2018;Farh et al. 2021;Methner et al. 2022).Consequently, S. fibuligera is increasingly recognized for its diverse adaptability and unique characteristics.
The mitochondrial genome of eukaryotes is indispensable for the regulation of growth and development, sustaining homeostasis and enabling the cell to react to the environment (Ernster and Schatz 1981;McBride et al. 2006;Murphy 2009).It has been suggested that the mitochondrial genome is a useful resource for examining fungal phylogeny (Xu and Wang 2015;Li et al. 2022aLi et al. , 2022bLi et al. , 2023a;;Gao et al. 2024).To date, two mitochondrial genomes from the Saccharomycopsis genus have been sequenced (Wolters et al. 2023).However, the mitochondrial genome characteristics of S. fibuligera have not been revealed.In the present study, we obtained the complete mitochondrial genome of S. fibuligera for the first time through next-generation sequencing technology, which has improved our understanding of the characteristics and evolution of the mitochondrial genome in the Saccharomycopsis genus.

Sample collection
In 2023, a specimen of S. fibuligera was isolated from a wine fermentation system in Luzhou (E 105.40 � , N 28.91 � ), Sichuan, China.Morphological and ITS rRNA sequencing were used to identify the specimens, which were then deposited at the Culture Collection Center of Luzhou Vocational and Technical College (contact person: Yue Deng; email: 157317724@qq.com) with voucher number Sfib1 (Figure 1).

Mitochondrial genome assembly and annotation
For DNA extraction from S. fibuligera, a fungal DNA extraction kit from Omega Bio-Tek (Norcross, GA, USA) was used.The NEBNextV R Ultra TM II DNA Library Prep Kit (NEB, Beijing, China) was then used for sequencing library preparation according to the manufacturer's instructions.Subsequently, the Illumina HiSeq 2500 Platform (Illumina, San Diego, CA, USA) was used for whole-genome sequencing.To guarantee the accuracy of the data, ngsShoRT (Chen et al. 2014) was used to filter out low-quality sequences, and AdapterRemoval v2 (Schubert et al. 2016) was used to remove adapter reads.The mitochondrial genome of S. fibuligera was de novo assembled using version 4.3.3 of NOVOPlasty, with a k-mer size of 31 (Dierckxsens et al. 2017).The mitochondrial genome was annotated in accordance with previously described methods (Li et al. 2019(Li et al. , 2020b(Li et al. , 2023b)), which involved the use of the MFannot tool (Lang et al. 2023) and MITOS (Bernt et al. 2013).By using the NCBI Open Reading Frame Finder, we can predict or modify protein-coding genes (PCGs) or open reading frames (ORFs) that are longer than 100 amino acids (Wu et al. 2017).Annotation of the functions of PCGs or ORFs was accomplished through BLASTP searches against the NCBI nonredundant protein sequence database (Bleasby and Wootton 1990).Exon and intron boundaries of PCGs were  accurately identified with the help of exonerate version 2.2 (Slater and Birney 2005).Using tRNAscan-SE v1.3.1, we confirmed the presence of tRNA genes in the S. fibuligera mitochondrial genome (Lowe and Chan 2016).OGDraw v1.2 was used to generate a graphical representation of the mitochondrial genome (Lohse et al. 2013).The structures of introncontaining genes were visualized using the PMGmap online web (http://www.1kmpg.cn/pmgmap,Supplementary Figure 1) (Zhang et al. 2024).

Discussion and conclusion
By utilizing the mitochondrial genome, we can gain a more comprehensive understanding of the phylogenetic relationships between species (Zhang et al. 2020;Ren et al. 2021;Zhang et al. 2022;2023).The absence of a mitochondrial reference genome for S. fibuligera impedes the application of the mitochondrial genome for classifying and investigating the phylogenetic relationships of Saccharomycopsidaceae fungi.In this study, we acquired the full mitochondrial genome of S. fibuligera.It was 57,302 bp in length, with a GC content of 24.40%.This genome included 14 core protein-coding genes (PCGs), 3 independent ORFs, 21 intronic ORFs, 25 tRNAs, and 2 rRNA genes.The S. fibuligera mitochondrial genome is three times smaller than the mitochondrial genome of S. malanga.By employing the BI phylogenetic inference method, we were able to construct phylogenetic trees for 24 Saccharomycotina fungi, with strong support for major clades (Shen et al. 2018;Groenewald et al. 2023); this demonstrated that S. fibuligera has a close relationship with S. capsularis.This study provides valuable information that is indispensable for the identification and recognition of Saccharomycopsis species, thus increasing our understanding of mitochondrial evolution and the varieties of Saccharomycopsidaceae fungi.

Figure 1 .
Figure 1. Isolation of the brewing yeast Saccharomycopsis fibuligera.This fungus was cultured on PDA media (200 g/L potato, 20 g/L glucose, and 20 g/L agar) at 28 � C for 3 days.A photo of the species was taken by Yue Deng.

Figure 2 .
Figure 2. Circular mitochondrial genome map of Saccharomycopsis fibuligera.Colored blocks outside each ring indicate that the genes are on the direct strand, while colored blocks within the ring indicate that the genes are located on the reverse strand.The inner grayscale bar graph shows the GC content of the mitochondrial sequences.The circle inside the GC content graph marks the 50% threshold.